Molecular diagnostics aims at the rapid detection of minute amounts of pathogens (typically bacteria) in samples such as blood. However, blood is a complex matrix and comprises white blood cells (leukocytes) for the adaptive immune system, red blood cells (erythrocytes) for oxygen transport, and platelets (thrombocytes) for wound healing. This composition complicates the direct detection of pathogens in samples such as whole blood, which contain a high amount of cellular material.
Classical detection methods comprise the growth of bacteria on selective media and/or media containing indicators. Typically such assays require a cultivation step of at least 1 or 2 days before identification of the bacteria can take place.
For PCR based methods the amount of bacteria in a fresh blood sample is theoretically high enough to be detected without further cultivation of the bacteria present within such sample. However, to allow an early detection of minute amounts of bacteria, large volumes of blood are required. The high amount of DNA in especially white blood cells dramatically increases the background in DNA based detection methods. Also the presence of heme from hemoglobin strongly decreases the activity of DNA polymerase. A microliter of human blood contains about 4,000 to 11,000 white blood cells and about 150,000 to 400,000 platelets. The concentration of DNA in blood is between 30 and 60 μg/ml. It is extremely challenging to detect the presence of about 10 to 100,000 of a bacterial species in a volume of 10 ml of whole blood.
The high amounts of DNA of the white blood cells may give rise to non relevant PCR products, or may scavenge the primers designed for the detection of bacterial DNA. This necessitates a thorough DNA purification and separation of eukaryotic DNA before the bacterial DNA can be detected via PCR or other methods.
Apart from interfering with the PCR reaction itself, the amount of mammalian DNA increases the viscosity of a sample. In addition, proteins and membranes from the lysed mammalian cells form complexes which prevent the filtration of a sample. This is particularly a problem for miniaturized devices. Further dilution of the large sample volume results in unacceptable long manipulation steps.
For the above reasons, methods to remove human DNA from a blood sample are accordingly required.
Methods to specifically assay bacterial DNA in the presence of mammalian DNA are known. Looxters™ from the company SIRSLab uses a method to enrich methylated DNA from a sample. As bacterial DNA is strongly methylated, this approach results in an enrichment of bacterial DNA. Molysis™ from the company Molzym, uses chaotropic agents and detergents to lyse selectively mammalian cells. This lysis step is followed by a digest with a DNAse which is not affected by this chaotropic agent/detergent. Alternative approaches such as commercialized by Roche (Septifast™) rely on PCR primer pairs which are specifically designed to prevent aspecific binding to human DNA and amplification of human DNA.
U.S. Pat. No. 6,803,208 describes a method wherein a highly diluted suspension of blood platelets doped with bacteria is lysed at 37° C. for 15 minutes, whereafter it is possible to filter a small amount of the lysed sample over a 0.4 μm filter for visual inspection of the bacteria which are retained on the filter. This method however does not allow to process large volumes of sample at ambient temperatures.
The non-published international patent application PCT/IB2010/055628 by Koninklijke Philips Electronics N.V. discloses a method for selective lysis of eukaryotic cells within a sample containing or suspected to contain micro-organisms, wherein a non-ionic detergent such as Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether) and a buffer is added to a sample comprising eukaryotic cells to obtain a solution having a pH value of at least 9.5, and incubating said solution for a time period sufficiently long enough to lyse the eukaryotic cells. This method permits processing of blood samples having a volume of 5 ml by lysing the white and red blood cells in the sample, degrading the blood cell DNA while pathogenic micro-organisms remain intact, and can subsequently be enriched by centrifugation or filtration.
In his article “Interactions of surfactants with lipid membranes” (Quarterly Reviews of Biophysics 41 (2008), pages 205-264), H. Heerklotz discusses the hypothetical molecular mechanism of selective lysis of mammalian cells, and hypothesizes that said selective lysis depends on different steps. First, the surfactant ensures lysis of the white and red blood cells. In order to achieve this, the surfactant needs to be inserted in the outer layer of the cell membrane. In a second step, the surfactant will perform a so-called flip-flop and is transferred to the inner layer of the cell membrane. Once a sufficient amount of surfactant is present in the inner cell membrane and the outer cell membrane, the cell will be lysed. Non-ionic surfactants such as Triton X-100 were found to be well suited for cell lysis as they perform above-mentioned steps within a time frame of several hundred milliseconds. In contrast, SDS requires 10 to 30 s for its insertion into PC vesicles. In addition, it is reported that surfactants with larger or charged head groups may require hours or days to cross the membrane, as was shown for SDS at room temperature.
This hypothesis may explain why ionic surfactants are not suitable for obtaining fast lysis of mammalian cells as has been described in scientific literature (see Heerklotz, H.). Surfactants comprising a large, bulky or charged hydrophilic group such as Tween®, ionic surfactants and Tritons having a long PEG chain are slow at the flip-flop movement and thus not suitable to obtain rapid cell lysis. In addition, surfactants having a very hydrophobic character such as Brij® 35 or Triton X-45 will encounter difficulties in their initial insertion into the cell membrane. Ionic surfactants are considered not suitable for obtaining fast lysis of mammalian cells, because their charged hydrophilic group cannot perform the flip-flop transfer easily due to the presence of the charged hydrophilic group which has to pass the lipophilic membrane.
In contrast to the scientific knowledge, it has surprisingly been found that an ionic surfactant can be utilized for selective lysis of white and red blood cells while keeping microbial pathogens intact when said ionic surfactant is used in combination with high pH. Thus, in a first aspect, the present invention provides a method for selective lysis of eukaryotic cells within a sample containing or suspected to contain micro-organisms. In a second aspect, the present invention provides a kit-of-parts for performing the method for selective lysis of eukaryotic cells within a sample containing or suspected to contain micro-organisms. In a further aspect, the present invention provides a device for detecting micro-organisms in a sample containing eukaryotic cells.